ORIGINAL ARTICLE

Cell Research (2006)16: 887-894
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Interaction of non-adherent suspended neutrophils to complement opsonized pathogens: a new assay using optical traps

Tomoko Suzuki, Masaru Yanai, Hiroshi Kubo, Akio Kanda, Hidetada Sasaki, James P Butler

¹Department of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, Sendai 980-8574, Japan; 2Physiology Program, Harvard School of Public Health, Boston, MA 02115, USA

Correspondence: Hiroshi Kubo Tel: +81-22-717-7182; Fax: +81-22-717-7186; E-mail: hkubo@geriat.med.tohoku.ac.jp Received 27 June 2006; revised 17 September 2006; accepted 21 September 2006; published online 24 October 2006

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Phagocytosis of opsonized pathogens by circulating non-adherent neutrophils is an essential step in host defense, which when overwhelmed contributes to sepsis. To investigate the role played by ligation of complement receptors CR3 and CR4 in non-adherent neutrophils, we designed a novel assay system utilizing dual optical traps, respectively, holding a suspended unactivated cell and presenting a specific ligand-coated bead to the cell surface. We chose anti-CD18 as an example ligand, mimicking the bacterial opsonizing complement fragment iC3b. Presentation of anti-CD18-coated beads elicited both pseudopodial protrusion and subsequent phagocytosis. This is in sharp contrast to previously reported responses of adherent neutrophils, which phagocytize opsonized particles without pseudopod formation. We used this same new assay to probe actomyosin pathways in the neutrophil¡¯s pseudopodial and phagocytic response. Disruption of actin or inhibition of myosin light-chain kinase dose-dependently reduced pseudopod formation and phagocytosis rates. In summary, i) the new dual trap assay can be used to study the responses of suspended neutrophils to a variety of ligands, and ii) in a first application of this technique, we found that local ligation of CR3/4 in unactivated neutrophils in suspension induces pseudopod formation and phagocytosis at that site, and that these events occur via an actomyosindependent pathway.

Cell Research (2006) 16:887-894. doi:10.1038/sj.cr.7310103; published online 24 October 2006

Keywords: neutrophil, phagocytosis, pseudopod formation, actomyosin, complement, CR3/4 receptor